Monoclonal antibodies have in recent years become successful therapeutic molecules, in particular for the treatment of cancer. Bispecific antibodies may further be able to increase the potency and efficacy of monoclonal antibody therapies, e.g. they could be used to direct a drug or toxic compound to target cells, to redirect effector mechanisms to disease-associated sites or to increase specificity for tumor cells, for example by binding to a combination of target molecules that is exclusively found on tumor cells. Furthermore, by combining the specificity of two monoclonal antibodies in one, bispecific antibodies could potentially engage a greater array of mechanisms of action c.q. their combined mechanisms of actions.
Different formats and uses of bispecific antibodies have recently been reviewed by Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276. One of the major obstacles in the development of bispecific antibodies has been the difficulty of producing the material in sufficient quality and quantity by traditional technologies, such as the hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649). Co-expression in a host cell of two antibodies, consisting of different heavy and light chains, leads to a mixture of possible antibody products in addition to the desired bispecific antibody.
Several strategies have been described to favor the formation of a heterodimeric, i.e. bispecific, product upon co-expression of different antibody constructs.
Lindhofer et al. (1995 J Immunol 155:219) disclose preferential species-restricted heavy/light chain pairing in rat/mouse quadromas.
A technique for formation of bispecific antibodies is the so-called “knob-into-hole” strategy (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and WO 2009089004 (Amgen) describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the heavy chain constant domain 3 (CH3), CH3-CH3 interfaces in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. WO2007110205 (Merck) describes yet another strategy, wherein differences between IgA and IgG CH3 domains are exploited to promote heterodimerization.
Dall'Acqua et al. (1998 Biochemistry 37:9266) have identified five energetically key amino-acid residues (366, 368, 405, 407 and 409) that are involved in the CH3-CH3 contact at the interface of a CH3 homodimer.
WO 2008119353 (Genmab) describes an ex vivo method for the generation of an antibody.
WO 11/131746 (Genmab) discloses heterodimeric antibody Fc-containing proteins and methods for production thereof.
The present invention relates to a method for production of heterodimeric proteins, such as stable IgG1 bispecific antibodies, wherein said method is particularly suitable for large-scale production of stable heterodimeric proteins wherein disulfide bonds are re-oxidized. By introduction of asymmetrical mutations in the CH3 domains of the homodimers, the Fab-arm exchange reaction can be forced to become directional due to complementarity of the CH3 domains, and thereby yield highly stable heterodimeric proteins.